Translational process engineering for tissue engineered hollow organ advanced therapy investigational medicinal products A thesis submitted to University College London for the degree of Doctor of Engineering by Toby Joseph Proctor September 2018 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London STATEMENT OF ORIGINALITY I, Toby Joseph Proctor, confirm that the work presented in this thesis is my own. Where information has been derived from other sources or through a collaborative means, I confirm that this has been indicated in the thesis. Print: Toby Joseph Proctor Date: September 2018 Page 2 of 257 ABSTRACT Tissue engineering has experienced increasing exposure and translational success in recent times, with tissue engineered products accounting for more than a quarter of approved advanced therapy medicinal products within Europe. Hollow organs represent a key target for developing novel therapies, typified by the recent success reported for tissue engineered hemilaryngeal replacements in a preclinical study. This thesis investigates the translational process engineering required to progress from a preclinical, good laboratory practice (GLP) process, to one that is compliant with good manufacturing practice (GMP) guidelines and suitable for clinical manufacture. A GLP decellularisation protocol was translated to conform to GMP-guidelines by modifying the existing standard operating procedure, and adapting an off-the-shelf bioreactor to form a closed-system for aseptic processing. The process was successfully validated for aseptic operation and decellularisation efficacy evaluated, relative to the preclinical process. The decellularised, human hemilarynx scaffolds produced were demonstrated to support bone marrow mesenchymal stromal cells (BM-MSCs) up to product release. A bespoke, modular bioreactor was designed and fabricated to enable manufacture of hemilarynges at scale. The bioreactor was successfully validated for aseptic use, whilst biocompatibility testing indicated no preclusion to use with BM-MSCs or epithelial cells. Proof-of-principle data supported the concept of epithelial sheet production inside the bioreactor, utilising a sheet-specific cassette. The bioreactor was retrospectively adapted to enable closed-system decellularisation processing of a third tissue-type, juvenile oesophagus. Acellular scaffold biomolecular composition and biomechanics were characterised, preceding implantation in a large- animal model. A second, bespoke bioreactor was designed, manufactured and employed to improve the manufacturing process. The combined human larynx data supported the award of a clinical trial authorisation, whilst the oesophageal work is now transitioning to a pivotal animal study. These findings support the application of bespoke bioreactor systems in process closure and translation towards robust, regulatory compliant, manufacturing processes for tissue engineered products. Page 3 of 257 IMPACT STATEMENT The research presented hereafter has, already, exhibited an impact outside of the originally intended thesis framework. The data presented in Chapter 2.2 and Chapter 4 have been submitted to the Medicines and Healthcare products Regulatory Agency as part of two investigational medicinal product dossiers, which have subsequently resulted in the award of clinical trial authorisations (EudraCT: 2013-004359-18 and EudraCT: 2015-002108-10). The data generated by these trials will provide information on the safety and efficacy of the respective advanced therapy investigational medicinal products, providing a foundation for subsequent pivotal studies. Should the therapies eventually receive marketing authorisation, they will provide a solution to the existing unmet clinical need associated with their respective indications. Additionally, the modular bioreactor described in Chapter 4 has been recently employed in the manufacture of a tissue engineered tracheal replacement for a paediatric patient, as an unlicensed medicine for human use, by the Centre for Cell, Gene and Tissue Therapeutics (MHRA MIA(IMP) 11149). The patient has, at time of print, experienced a gross improvement in quality of life. Page 4 of 257 ACKNOWLEDGEMENTS I would like to take the opportunity to thank all those that have contributed to my doctoral experience throughout my studies, and to acknowledge the funding provided by the Engineering and Physical Sciences Research Council that supported the work documented herein. I would like to thank Dr Farlan Veraitch for his enthusiasm, support, and insight during my time with the Department of Biochemical Engineering. I would also like to thank Professor Mark Lowdell, in whose group I have spent the last five years, for bringing me into the world of translational medicine. The experiences that I have been exposed to during my time with the Centre for Cell, Gene and Tissue Therapeutics are beyond what most doctoral students could ever dream of. He has taught me that, whilst Heath Robinson may have been a visionary, simplicity usually does the trick. Erich Herrmann, thank you for your patience with a former biologist trying to build a box. Without your input I would not be heading down the career path on which I now find myself. Thanks, also, to Dan Fernandez, Phil Sands and Denzil Booth for bioreactor fabrication. Thank you to the entire team at the Royal Free, the CCGTT is as much the people as it is anything else. Great thanks to John Yaxley for keeping us all as close to the straight and narrow as could be reasonably expected. Angela, thank you for everything epithelial, and for accepting responsibility as head angry-email-sender. Xenia, thank you for all the emergency reagent changes and for kindly pointing out that my staining was rubbish. Special thanks and gratitude has to go to Carla, without whom I would not have survived the last five years. Your guidance gave me a true, first-hand insight into what GMP really is. Although we may never make it as song writers, our cleanroom karaoke renditions will stay with me forever. I would like to express my love and thanks to Sarah Nicholson, whom has shared and witnessed the entirety of my university education. Thank you for putting it all in perspective. Finally, I would like to thank my parents for raising me to be who I am today. Dad, thank you for introducing me to the wonderful world of seals. One day we will just use grease. And mum, look at how far you have managed to get me beyond reading ‘the’. It is beyond belief. Thank you both for always supporting me. Page 5 of 257 TABLE OF CONTENTS Statement of Originality .................................................................................................. 2 Abstract .......................................................................................................................... 3 Impact statement ............................................................................................................ 4 Acknowledgements ........................................................................................................ 5 List of figures ................................................................................................................ 10 List of tables ................................................................................................................. 12 Abbreviations ................................................................................................................ 13 1 Introduction ........................................................................................................... 15 1.1 Regenerative medicine and tissue engineering ............................................. 15 1.2 European regulation of advanced therapies .................................................. 18 1.2.1 Advanced therapy medicinal product classification ................................ 18 1.2.2 The current European ATMP landscape ................................................. 21 1.2.3 Tissue Engineered ATMPs ..................................................................... 21 1.3 Hollow organ targets for ATMP development ................................................ 23 1.3.1 Larynx ..................................................................................................... 23 1.3.2 Tissue engineering approaches to laryngeal regeneration ..................... 27 1.3.3 Oesophagus ........................................................................................... 28 1.3.4 Tissue engineering approaches to oesophageal regeneration ............... 29 1.5 Thesis aims .................................................................................................... 31 2 Materials and methods .......................................................................................... 32 2.1 General materials and methods ..................................................................... 32 2.1.1 Tissue procurement and preparation ...................................................... 32 2.1.2 Cell isolation and expansion ................................................................... 34 2.1.3 Biomolecular quantitative analysis .......................................................... 36 2.1.4 Qualitative tissue analysis....................................................................... 37 2.1.5 Statistical analysis ................................................................................... 38 2.2 Chapter specific materials and methods – Chapter 3 ...................................